Anti gpr87

Western blot was performed using anti-GPR87 (Abcam), anti-p-IκBα, IκBα and anti-p-IKKβ, IKKβ, anti-p65, anti-p84 antibodies (Cell Signaling Technology). The membranes were stripped and re-probed with an anti-α-tubulin antibody (Sigma) as a loading control.

Whole-cell lysates were prepared using RIPA buffer (iNtRON Biotechnology, Seongnam, Korea) with a protease inhibitor cocktail (Roche, Basel, Switzerland). Total protein samples were quantified using the Pierce BCA Protein Assay Kit (Thermo, Waltham, MA, USA). Equal amounts of protein lysates were separated on 8–16% Bis-Tris protein gels (Invitrogen, Waltham, MA, USA), transferred to PVDF membranes (Millipore, Burlington, MA, USA), and blocked with 5% skim milk (BD Biosciences, NJ, USA). The following primary antibodies were used: anti-GPR87 (Abcam, Cambridge, UK), anti-N-cadherin, anti-ZEB1, anti-E-cadherin, anti-Claudin, anti-phospho-AKT, anti-AKT, anti-phospho-eNOS, anti-eNOS and horseradish peroxidase (HRP)-conjugated anti-β-actin, (Cell Signaling, Danvers, MA, USA). An HRP-conjugated anti-mouse IgG antibody (Bio-Rad, Hercules, CA, USA) was used as the secondary antibody. Specific bands were detected using the WEST-ZOL plus Western Blot Detection System (iNtRON Biotechnology, Seongnam, Korea).